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Objective: To prepare a fucoidan-modified phase-transitional contrast agent (FPCA) and to evaluate its in vitro capabilities for ultrasound imaging and targeting of hepatoma cells., Methods: Nano-liposomes encapsulated with perfluoropentane were prepared using thin-film hydration and ultrasonic emulsification methods. Then, FPCA nanoparticles were prepared through chemical grafting of fucoidan and the characterization of their physical and chemical properties was performed. After applying external stimuli of heating with hot water bath and microwave irradiation, the phase-transition status of FPCA was observed with microscope. The imaging abilities of phase-transited FPCA on two-dimensional ultrasound and contrast-enhanced ultrasound were observed with ultrasonic diagnostic instrument. The ability of FPCA to target at hepatoma cells was evaluated and verified with fluorescence confocal observation and flow cytometry analysis., Results: The FPCA prepared in the study had an average diameter of (222.1±32.5) nm, displaying spherical appearance, good dispersion, good stability, and good biocompatibility. The phase-transition of FPCA was induced by both heating with hot water bath and microwave irradiation. For phase transition, the optimal temperature was found to be 50 ℃ and the preferred microwave power was 1.5 W/cm 2 . Moreover, after phase transition, FPCA showed significant imaging enhancement on both two-dimensional ultrasonography and contrast-enhanced ultrasonography. Through fluorescein isothiocyanate (FITC) labeling, FPCA could specifically bind with hepatoma cells at a high binding rate of (96.19±1.62)%, while it rarely bound with normal liver cells, showing a binding rate of less than 10%., Conclusion: A new type of phase-transitional ultrasound contrast agent with good stability and biocompatibility was successfully prepared. It not only could enhance ultrasound imaging through phase transition, but also had specific active hepatoma cell-targeting properties., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

In order to prepare angiopep-2 modified fluorescein isothiocyanate-labeled neurotoxin nanoparticles( ANG-NPs/FITCNT),emulsion/solvent evaporation method was used with m PEG-PLA and ANG-PEG-PLA( in proper proportions) as carriers and with FITC-NT as drug. With particle size and encapsulation efficiency as comprehensive indexes,the effects of different ultrasound power and ultrasound time combinations on the process were investigated. The in vitro release characteristics of nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were investigated by dialysis method. The results indicated that the optimum process for preparing ANG-NPs/FITC-NT was as follows: ultrasonic power 90 W,ultrasonic time 30 s. In such optimal process,ANG-NPs/FITC-NT were well-shaped under the transmission electron microscope,with an average particle size of( 123. 9±0. 5) nm,Zeta potential of(-10. 5±0. 5) m V,encapsulation efficiency of( 68. 1±0. 4) %,and the drug loading of( 0. 82±0. 01) %. The in vitro drug release profiles of the nanoparticles in PBS buffer at p H 7. 4 and p H 6. 5 were both consistent with Ritger-Peppas equation,ln Q = 0. 508 8 lnt-2. 285 0,r = 0. 961 5( p H 7. 4) and ln Q= 0. 449 9 lnt-1. 855 3,r = 0. 970 3( p H 6. 5),respectively. The experiment results proved that the nanoparticles prepared by emulsion/solvent evaporation method had uniform particle size,high encapsulation efficiency and in vitro sustained release characteristic,which might be a potential carrier for NT intracerebral drug delivery.

Objective: To investigate the application of the optical magnetic bimodal molecular probe Gd-DO3A-ethylthiouret-fluorescein isothiocyanate (Gd -DO3A-EA-FITC) in brain tissue imaging and brain interstitial space (ISS)., Methods: In the study, 24 male SD rats were randomly divided into 3 groups, including magnetic probe group (n=6), optical probe group (n=6) and optical magnetic bimodal probe group (n=12), then the optical magnetic bimodal probe group was divided equally into magnetic probe subgroup (n=6) and optical probe subgroup (n=6). Referencing the brain stereotaxic atlas, the coronal globus pallidus as center level, the probes including gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA), fluorescein isothiocyanate (FITC) and Gd-DO3A-EA-FITC of 2 μL (10 mmol/L) were injected into the caudate nucleus respectively, magnetic resonance imaging (MRI) was performed in the magnetic probe group and magnetic probe subgroup to image the dynamic diffusion and distribution of the probes in the brain ISS, a self-developed brain ISS image processing system was used to measure the diffusion coefficient, clearance, volume fraction and half-time in these two groups. Laser scanning confocal microscope (LSCM) was performed in vitro in the optical probe group and optical probe subgroup for fluorescence imaging at the time points 2 hours after the injection of the probe, and the distribution in the oblique sagittal slice was compared with the result of the first two groups., Results: For the magnetic probe group and magnetic probe subgroup, there were the same imaging results between the probes of Gd-DTPA and Gd-DO3A-EA-FITC. The diffusion parameters of Gd-DTPA and Gd-DO3A-EA-FITC were as follows: the average diffusion coefficients [(3.31±0.11)×10 -4 mm 2 /s vs. (3.37±0.15)×10 -4 mm 2 /s, t=0.942, P=0.360], the clearance [(3.04±0.37) mmol/L vs. (2.90±0.51) mmol/L, t=0.640, P=0.531], the volume fractions (17.18%±0.14% vs. 17.31%±0.15%, t=1.961, P=0.068), the half-time [(86.58±3.31) min vs. (84.61±2.38) min, t=1.412, P=0.177], the diffusion areas [(23.25±0.68) mm 2 vs. (22.71±1.00) mm 2 , t=1.100, P=0.297]. The statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant. Moreover, for the optical probe group and optical probe subgroup, the diffusion area of Gd-DO3A-EA-FITC [(22.61±1.16) mm 2 ] was slightly larger than that of FITC [(22.10±1.29) mm 2 ], the statistical analysis of each brain was made by t test, and the diffusion parameters were not statistically significant (t=0.713, P=0.492)., Conclusion: Gd-DO3A-EA-FITC shows the same imaging results as the traditional GD-DTPA, and it can be used in measuring brain ISS.

An experimental method of protein separation by capillary electrophoresis coupled with light-emitting diode induced fluorescence detection (CE-LED-IF) was established. The fluorescein isothiocyanate (FITC) was used as fluorescent dye in this method. An optical fiber was inserted into the separation capillary and was suited right at the detection window which was used to transmit the excitation light of LED. The efficiency of the light of LED and the detection limits of FITC was improved. The difference of serum proteins between the patients with cirrhosis and normal people was detected by this method. A series of effects on CE were studied, such as the wavelength of the light source and filter, the concentration and pH of the running buffer and the separation voltage. Then the optimized method was established for the determination of the serum proteins. The repeatability of CE-LED-IF was tested and the RSDs of migration time and peak area were 1. 14% and 3. 23% , respectively. The results showed that there were two specific peaks in cirrhosis serum. The differences between the patients with cirrhosis and normal people having statistical significance indicated that these proteins might be the potential biomarkers for cirrhosis diagnosis. The developed CE-LED-IF method proved suitable for the separation of serum proteins with good repeatability and supportable for the clinical diagnosis of cirrhosis.

An in vitro detection method of the gastrointestinal absorption of Pilose Antler protein was established for mixed protein activity. Five bands of protein with molecular weight of 17.8-160 kD derived from the Pilose Antler were extracted and sufficiently labeled with FITC (FITC-PE). The stability and variation of FITC-PE in gastrointestinal circumstances were detected by native polyacrylamide gel electrophoresis and confocal laser scanning microscope. Results showed that the main component of FITC-PE kept invariant after being reacted with artificial gastric fluid and artificial intestinal fluid. The fluorescence signal was detected 20 min after administration in the valgus intestinal purse experiment, and three kinds of protein, with molecular weight of 45, 25, and 17.8 kD, were detected in the mixture of absorbent protein. The research laid the foundation for the further in vivo study of Pilose Antler protein. Meanwhile, it would be an in vitro screening method for the absorption, distribution and metabolism of mixed protein from traditional Chinese medicine.

In the present work, the amount and the activity of the goat anti-human IgG, to bind the human IgG labelled with fluorescein isothiocyanate (FITC), immobilized on silicon surfaces modified with APTES and APTES-Glu, respectively, were studied using the fluorescence spectrum imaging (FSI), the results of which were compared with that of ellipsometry. It is shown that the amount of the human IgG labeled with FITC on APTES-Glu measured using FSI is 2.8 times higher than that on APTES, which is nearly coincident with the 2.2 times obtained using ellipsometry, showing that the activity of the goat anti-human IgG on APTES-Glu is higher than that on APTES. It is reasoned that the FSI is used in the fluorescence immunoassay the for measurement of quasi-quantification or quantification.

Aim: To investigate the absorption and distribution of recombinant hirudin-2 (rHV2) in the GI tract in rats after oral administration., Methods: Using HPLC, fluorescence spectrophotometry and confocal laser scanning microscopy to measure the amount of intact rHV2 absorbed into gastrointestinal mucosa and blood., Results: HPLC spectrum showed that there were intact rHV2 molecules in plasma after 1 h of oral administration. After oral administration for 3 h, 1.2%-26.8% of fluorescence was found in the GI tract in rats. Chromatographic analysis showed that 2.27%-38.75% of fluorescence recovered from the GI tract luminal contents and mucosa was eluted at the void volume of a Sephadex G-25 column. Microscopic examination showed that FITC-rHV2 was taken up throughout the whole small intestine but the ileum appeared to be a preferred site for FITC-rHV2 transport in rats., Conclusion: rHV2 may partially survive in the GI lumen and subsequently absorbed in active form by gastrointestinal tract.

Aim: To establish a simple and effective method for RBCs labeling and survival assays, and the qualities of rabbit RBCs preserved in GMA solution at 4 degrees C were verified., Methods: The bloods were taken through the ear arteries of the rabbits. The RBCs were labeled by fluorescein isothiocyanate (FITC), and were reinjected to the same rabbit through ear veins. The percentage of FITC labeled RBCs was assayed by FACS at a series of times after injection. The SAS software was employed to analyze the data and establish the regression equations. The 24-hour recovery and the half-life span of the labeled RBCs were calculated according to the equations., Results: The 24-hour recovery and the half-life span of the labeled RBCs in the control group were 93.76% +/- 5.40% and 22.50% +/- 4.37 days respectively, which was in agreement with the previous papers. The 24-hour recovery and the half-life span of the labeled RBCs in the GMA group were 89.13% +/- 7.10% and 11.41% +/- 1.63 days respectively, which was coincident with the infusion conditions., Conclusion: Compared with other methods of RBCs labeling in vivo, FITC labeling was thought to be easier and cheaper to use, which could facilitate the analysis of the biological character of the labeled cells, and could be used to trace the fate of labeled cells.

The properties of room temperature phosphorescence (RTP) of the labeled goat anti-human antibody(with fluorescein isothiocynate GAHAb-FITC) and labeled rabit anti-goat antibody(RAGAb-FITC) with FITC in different dilution ratios on many kinds of solid substrates were studied in detail. It is found that on the polyamide membrane, only using Pb(Ac)2 as a heavy atom perturber, both the labeled antibodies and their antigen-antibody conjugated compound with the human immunoglobulin G remained excellent RTP properties of isothiocyanate fluorescein, lambda ex(max)/lambda em(max) = 525/650 nm, the relation of RTP intensity to the concentration was linear in a certain range. The conditions for immunological reaction and RTP emission were optimized. The result means that it is possible to develop a new immunoassay named as solid substrate room temperature phosphorescence immunoassay (SS-RTP-IA), which combines both the specificity of immunological reaction and high sensitivity of luminescence analysis.

Objective: To explore the characteristics of the combination of LPS to the endothelium and endothelial injury., Methods: Myocardial microvascular endothelial cells (MMECs) were isolated and cultured to adhering state. Flow cytometry, laser confocal scanning microscope (LCSM) and image analysis were employed to evaluate the effects of different concentrations of LPS and different culturing time with the same concentration of LPS on the adhered MMECs, Results: (1) The OD values of MMECs increased progressively along with the prolongation of culturing time of MMECs with LPS and reached top level at 30 min. In addition, the increase of OD values was dependent on time. (2) The number of positive MMECs and the OD values increased after MMECs was cultured for 2 hours with LPS at concentrations ranging from 0.03125 similar 2.0000 g/L. The OD value reached peak level when LPS concentration was 0.2500 g/L, which implied that this concentration of LPS (0.25 g/L) was the optimal one for the combination of LPS with MMECs, and there was concentration saturation. (3) LPS could enter into the cytoplasm and nucleus of MMECs. (4) LPS could induce nucleic translocation and denucleation., Conclusion: (1) LPS could combine MMECs and enter into the nucleus without the presence of serum. (2) LPS could directly damage MMECs.

Eighty eight healthy Wistar rats were used in this study. Pseudomonas aeruginosa (serotype IX, labelled with fluorescein isothiocyanate) were fed in suspension (10(9)/ml, 1ml/100g body weight) following a 25-28% full-thickness burn created on the back of animals. The animals were sacrificed at eight different time phases from 1-72 hours after burns. The labelled bacteria were traced in internal organs, the quantitative bacterial cultures of visceral tissues and blood cultures were performed coincidently. The results showed that the labelled bacteria introduced into GI tract could be demonstrated from the internal organs and blood circulation.